Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions (sometimes referenced as features or spots) of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. The arrays, when exposed to a sample, will exhibit a binding pattern. This binding pattern can be observed, for example, by labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), and accurately observing the fluorescence pattern on the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated using either deposition of the previously obtained biopolymers or in situ synthesis methods. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different regions of the substrate to yield the completed array. Typical procedures known in the art for deposition of previously obtained polynucleotides, particularly DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution in one or more drop dispensers such as the tip of a pin or in an open capillary and, touch the pin or capillary to the surface of the substrate. Such a procedure is described in U.S. Pat. No. 5,807,522. When the fluid touches the surface, some of the fluid is transferred. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for many different sequences and, eventually, the desired array is formed. Alternatively, the DNA can be loaded into a drop dispenser in the form of an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere.
The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA) using phosphoramidite or other chemistry. Such in situ synthesis methods can be basically regarded as iterating the sequence of depositing droplets of: (a) a protected monomer onto predetermined locations on a substrate to link with either a suitably activated substrate surface (or with a previously deposited deprotected monomer); (b) deprotecting the deposited monomer so that it can now react with a subsequently deposited protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one cycle so that the different regions of the completed array will carry the different biopolymer sequences as desired in the completed array. One or more intermediate further steps may be required in each iteration, such as oxidation and washing steps.
It is important in such arrays that features actually be present, that they are put down accurately in the desired pattern, are of the correct size, and that the DNA is uniformly coated within the feature. However, in the conventional in situ methods for polynucleotide arrays, phosphoramidite nucleoside monomers are used. In order for the phosphoramidite group to link to a hydroxyl of a previously deposited deprotected polynucleotide monomer, it must first be activated usually by using a weak acid such as tetrazole. However, an activated phosphoramidite is highly reactive with moisture in the air. This leads to a reduction in deposited monomer available for reaction. Furthermore, since water tends to be adsorbed initially at the surface of a droplet which is being used in one cycle of forming a polynucleotide at a feature, the phosphoramidite concentration at the surface of the droplet will tend to be lowest. Consequently, the concentration of a completed probe polynucleotide at a feature of the array, tends to decrease from the center of a feature toward its perimeter. This leads to a decrease in the total signal that should be available when a target to which that polynucleotide hybridizes, is detected. Furthermore, since water vapor concentration the ambient atmosphere may vary, such signal may also vary from array to array, leading to inconsistency in absolute signal generated from different arrays of a batch when the same concentration of a target is encountered. The foregoing problems particularly exist where the phosphoramidite is mixed with activator and the mixture deposited as a droplet on the substrate, such as described in PCT publication WO98-41531. The foregoing reference also states that the activator can be deposited onto a previously deposited droplet containing the phosphoramidite. However, the potential for the activated phorphoramidite to react with moisture in the ambient atmosphere still exists. Furthermore, when one droplet is deposited on the other, there is no guarantee of efficient mixing such that the activated phosphoramidite will be evenly present at the substrate surface.
It would be desirable then, in the fabrication of arrays of biopolymers using biomonomers with a linking group which must be activated (such as a phosphoramidite), to provide a means by which potential reactivity of the activated biomonomer with an ambient atmosphere component (such as water vapor in air) can be kept low.